June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Molecular, physiological and morphological effects of DHDDS knockdown in photoreceptors of Drosophila
Author Affiliations & Notes
  • Liliana Mizrahi-Meissonnier
    Ophthalmology, Hadassah Medical Center, Jerusalem, Israel
  • Rachel Zaguri
    Faculty of Medicine, Hebrew University, Jerusalem, Israel
  • Elisheva Rhodes
    Faculty of Medicine, Hebrew University, Jerusalem, Israel
  • Vladimir Katanaev
    Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland
  • Baruch Minke
    Faculty of Medicine, Hebrew University, Jerusalem, Israel
  • Dror Sharon
    Ophthalmology, Hadassah Medical Center, Jerusalem, Israel
  • Footnotes
    Commercial Relationships Liliana Mizrahi-Meissonnier, None; Rachel Zaguri, None; Elisheva Rhodes, None; Vladimir Katanaev, None; Baruch Minke, None; Dror Sharon, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5441. doi:
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      Liliana Mizrahi-Meissonnier, Rachel Zaguri, Elisheva Rhodes, Vladimir Katanaev, Baruch Minke, Dror Sharon; Molecular, physiological and morphological effects of DHDDS knockdown in photoreceptors of Drosophila. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5441.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: In 2011, others and we reported on the identification of a founder mutation in the dehydrodolichyl diphosphate synthase (DHDDS) gene in Ashkenazi Jews with non-syndromic retinitis pigmentosa (RP). The DHDDS enzyme is known to be involved in dolichol synthesis, a lipid molecule that is important for various biological functions, including N-glycosylation and membrane fluidity. The fact that the patients do not suffer from other clinical problems is surprising since genetic defects in related enzymes cause a systemic phenotype with involvement of multiple organs. The purpose of the current study was to construct and analyze a Drosophila strain in which DHDDS expression is knocked-down in photoreceptors using RNAi.

Methods: The DHDDS-RNAi (CG10778-RNAi) Drosophila strain under the control of the GMR photoreceptor specific promoter was generated by crossing w-;P[UAS: CG10778-RNAi]; Sb+flies with a GMR-Gal4 strain to drive maximal suppression of CG10778-RNAi. Flies were raised under on a 12H:12H dark/light cycle, in complete dark or in 24H intense light in 25C and collected 1 and 7 days after eclosion. Electroretinogram (ERG) was recorded using standard techniques. The Prolonged Depolarizing Afterpotential (PDA) was used as an assay for in vivo electrophysiological measure of the fly rhodopsin level. Transmission electron microscopy (TEM) analysis was performed on fly eye. Western blot analysis was performed with several antibodies.

Results: Western blot analysis revealed that DHDDS expression was downregulated in the CG10778-RNAi fly photoreceptors. ERG analysis of CG10778-RNAi flies showed an abnormally small and short PDA indicating that rhodopsin (Rh1) level was drastically reduced in the transgenic fly, as verified directly by western blot analysis. TEM analysis revealed an abnormal retinal structure indicating an early-onset and severe photoreceptor degeneration. Interestingly, not all photoreceptors were equally affected by the degenerative process and developmentally-dependent specific subtypes were spared.

Conclusions: CG10778-RNAi Drosophila is an excellent model to study DHDDS function. We were able to show by molecular (Western blot analysis), physiological (ERG) and morphological (TEM) methods that DHDDS knockdown reduced the rhodopsin expression in Drosophila photoreceptors causing retinal degeneration.

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